Although several immunological reagents have been developed (ex: tumor infiltrating lymphocytes, antigen specific tumor vaccines etc.) during the last few years which can recognize and kill melanoma cells and have the potential of curing melanoma patients, most patients are resistant to this form of therapy. This resistance is probably dependent upon a complexity of variables affecting the balance between activation of the immune system and growth of tumor cells in vivo and our limited understanding of them. My work is focused on analyzing variables potentially affecting this balance. During the last year we have further addressed several issues related to this topic, specifically: 1. The quality and quantity of the immune response to peptide-based vaccination. Our data suggest that the precursor frequencies obtained using the current vaccination regimens are not comparable with the precursor frequency of T cell specific for exogenous pathogens during acute or chronic infections. We have shown that different schedules of vaccine administration could enhance the immune responses against cancer. In particular the number and frequency of vaccines might be a critical factor in increasing the frequency of number of tumor reactive T cells in the peripheral blood. We have also demonstrated that vaccine-elicited T cells reach and localize at tumor site. However, this is not sufficient for tumor rejection. Various tumor escape mechanisms were also explored during the last year but not lead was identified regarding what additional biological process is involved in immune rejection of cancer. Several technologies were implemented or developed in our laboratory such as preparation and utilization of soluble HLA/epitope tetramers, TaqMan-based real time PCR, intra-cellular FACS technology and development of a technique allowing to expand tumor/T cell pairs from fine needle aspirates of metastatic melanoma. Addressing this issue several reports were published during the last year. To increase the efficiency of our search for mechanisms leading to tumor regression in humans we have develop a new method that allows the utilization of material derived from fine needle aspirates of tumors to be studies in high throughput systems such as micro-arrays. Presently we are comparing, using this new method, the expression profile of gene expressed in lesions that did or did not respond to therapy.